In addition to its positive signaling function in the antigen presentation process, CD4 acts as the primary receptor for HIV-1. Contact between CD4 and the viral envelope leads to virus entry, but can also trigger apoptosis of uninfected CD4+ T-cells through a mechanism that is poorly understood. We show that Siva-1, a death domain-containing proapoptotic protein, associates with the cytoplasmic domain of CD4. This interaction is mediated by the cysteine-rich region found in the C-terminal part of the Siva-1 protein. Expression of Siva-1 specifically increases the susceptibility of both T-cell lines and unstimulated human primary CD4+ T-lymphocytes to CD4-mediated apoptosis triggered by the HIV-1 envelope, and results in activation of a caspase-dependent mitochondrial pathway. The same susceptibility is observed in T-cells expressing a truncated form of CD4 that is able to recruit Siva-1 but fails to associate with p56Lck, indicating that Siva-1 participates in a pathway independent of the p56Lck kinase activity. Altogether, these results suggest that Siva-1 might participate in the CD4-initiated signaling apoptotic pathway induced by the HIV-1 envelope in T-lymphoid cells.
"As of yet, published reports of a nuclear specific function for Siva are lacking. Most detailed investigations of Siva function have examined Siva in relation to transmembrane signalling molecules or mitochondrial-associated apoptosis regulators thought to be present in the cytoplasm, not in the nucleus [28,29,37,43,44,50,52-54]. In future studies, it will be interesting to look into whether Siva displays nucleus-specific activities such as direct binding to DNA or chromatin. "
[Show abstract][Hide abstract] ABSTRACT: Severe autoinflammatory diseases are associated with mutations in the Foxp3 locus in both mice and humans. Foxp3 is required for the development, function, and maintenance of regulatory T cells (Tregs), a subset of CD4 cells that suppress T cell activation and inflammatory processes. Siva is a pro-apoptotic gene that is expressed across a range of tissues, including CD4 T cells. Siva interacts with three tumor necrosis factor receptor (TNFR) family members that are constitutively expressed on Treg cells: CD27, GITR, and OX40.
Here we report a biophysical interaction between FOXP3 and Siva. We mapped the interaction domains to Siva's C-terminus and to a central region of FOXP3. We showed that Siva repressed IL-2 induction by suppressing IL-2 promoter activity during T cell activation. Siva-1's repressive effect on IL-2 gene expression appears to be mediated by inhibition of NFkappaB, whereas FOXP3 repressed both NFkappaB and NFAT activity.
In summary, our data suggest that both FOXP3 and Siva function as negative regulators of IL-2 gene expression in Treg cells, via suppression of NFAT by FOXP3 and of NFkappaB by both FOXP3 and Siva. Our work contributes evidence for Siva's role as a T cell signalling mediator in addition to its known pro-apoptotic function. Though further investigations are needed, evidence for the biophysical interaction between FOXP3 and Siva invites the possibility that Siva may be important for proper Treg cell function.
"Interaction mapping with truncated versions of both proteins revealed that the Cterminal RING domain of XIAP is sufficient for binding to the N-terminal, SAH and DDHR-containing domain of Siva1. Several interaction partners of Siva1 are known so far, including CD27, the CVB3 capsid protein VP2 and the peroxisomal membrane protein PMP22, which interact with the N-terminal part of Siva1, whereas the lysophosphatidic acid (LPA) receptor 2 and the cytoplasmatic domain of CD4 interact with the C-terminal part of Siva1 (Henke et al., 2000; Lin et al., 2007; Nestler et al., 2006; Prasad et al., 1997; Py et al., 2007). Furthermore, the SAH domain of Siva1 has been shown to interact with and inhibit the antiapoptotic protein Bcl-xL, thereby sensitizing cells to UVradiation-induced apoptosis (Xue et al., 2002). "
[Show abstract][Hide abstract] ABSTRACT: XIAP is known as a potent inhibitor of apoptosis, but in addition is involved in cellular signalling, including the NFkappaB, JNK and TGFbeta pathways. Our search for XIAP-interacting partners led us to Siva1, a proapoptotic protein that is known to play a role in T-cell apoptosis through a caspase-dependent mitochondrial pathway. The interaction sites between XIAP and Siva1 were mapped to the RING domain of XIAP and the N-terminal, SAH-containing and death-homology-region-containing domains of Siva1. Co-immunoprecipitation experiments showed that XIAP, Siva1 and TAK1 form a ternary complex in Jurkat T cells. Reporter-gene analysis revealed that Siva1 inhibits XIAP- and TAK1-TAB1-mediated NFkappaB activation. By contrast, Siva1 increased XIAP- and TNFalpha-mediated AP1 activity and prolonged TNFalpha-induced JNK activation, whereas knock down of Siva1 resulted in reduced JNK activation. This suggests that Siva1 differentially modulates signalling by JNK and NFkappaB and shifts the balance between these pathways towards enhanced JNK activation, a situation that promotes apoptosis. Ectopically expressed Siva1 increased caspase-3 activity, which was inhibited by XIAP in a ubiquitin-ligase-dependent manner. In line with this, Siva1 was lysine-48-linked polyubiquitylated by XIAP. Our findings suggest that, via physical interaction with XIAP and TAK1, Siva1 diminishes NFkappaB and enhances JNK activity to favour apoptosis.
"The vector for expression of the cytoplasmic domain of CD4 fused to LexA has been described . The DNA sequence coding for PLSCR1 was amplified by PCR from a cDNA library  and cloned into the pGAD3S2X plasmid (Clontech) to generate the pGAD-PLSCR1 vector for expression of PLSCR1 fused to the Gal4 activation domain (Gal4AD). "
[Show abstract][Hide abstract] ABSTRACT: Secretory leukocyte protease inhibitor (SLPI) is secreted by epithelial cells in all the mucosal fluids such as saliva, cervical mucus, as well in the seminal liquid. At the physiological concentrations found in saliva, SLPI has a specific antiviral activity against HIV-1 that is related to the perturbation of the virus entry process at a stage posterior to the interaction of the viral surface glycoprotein with the CD4 receptor. Here, we confirm that recombinant SLPI is able to inhibit HIV-1 infection of primary T lymphocytes, and show that SLPI can also inhibit the transfer of HIV-1 virions from primary monocyte-derived dendritic cells to autologous T lymphocytes. At the molecular level, we show that SLPI is a ligand for the phospholipid scramblase 1 (PLSCR1) and PLSCR4, membrane proteins that are involved in the regulation of the movements of phospholipids between the inner and outer leaflets of the plasma membrane. Interestingly, we reveal that PLSCR1 and PLSCR4 also interact directly with the CD4 receptor at the cell surface of T lymphocytes. We find that the same region of the cytoplasmic domain of PLSCR1 is involved in the binding to CD4 and SLPI. Since SLPI was able to disrupt the association between PLSCR1 and CD4, our data suggest that SLPI inhibits HIV-1 infection by modulating the interaction of the CD4 receptor with PLSCRs. These interactions may constitute new targets for antiviral intervention.
PLoS ONE 02/2009; 4(3):e5006. DOI:10.1371/journal.pone.0005006 · 3.23 Impact Factor
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